Hahn, Young S. and Grakoui, Arash and Rice, Charles M. and Strauss, Ellen G. and Strauss, James H. (1989) Mapping of RNA- temperature-sensitive mutants of Sindbis virus: complementation group F mutants have lesions in nsP4. Journal of Virology, 63 (3). pp. 1194-1202. ISSN 0022-538X. PMCID PMC247815. http://resolver.caltech.edu/CaltechAUTHORS:HAHjvir89b
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Temperature-sensitive (ts) mutants of Sindbis virus belonging to complementation group F, ts6, ts110, and ts118, are defective in RNA synthesis at the nonpermissive temperature. cDNA clones of these group F mutants, as well as of ts+ revertants, have been constructed. To assign the ts phenotype to a specific region in the viral genome, restriction fragments from the mutant cDNA clones were used to replace the corresponding regions of the full-length clone Toto1101 of Sindbis virus. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious transcripts, and the virus recovered was tested for temperature sensitivity. After the ts lesion of each mutant was mapped to a specific region of 400 to 800 nucleotides by this approach, this region of the cDNA clones of both the ts mutant and ts+ revertants was sequenced in order to determine the precise nucleotide change and amino acid substitution responsible for each mutation. Rescued mutants, which have a uniform background except for one or two defined changes, were examined for viral RNA synthesis and complementation to show that the phenotypes observed were the result of the mutations mapped. ts6 and ts110 had a single base substitution in nsP4, resulting in replacement of Gly by Glu at position 153 or position 324, respectively. It is of interest that nsP4 contains the Gly-Asp-Asp motif characteristic of a number of viral replicases, and this, together with the fact that all RNA synthesis in ts6-infected cells and, to a lesser extent, in ts110-infected cells shut off when the cells were shifted from a permissive to a nonpermissive temperature, suggests that nsP4 is the virus polymerase. ts118 was a double mutant. It contained a single base substitution in nsP2, resulting in replacement of Val by Ala at position 425 that resulted in the formation of minute plaques, but not in a reduction in the plaque number at the nonpermissive condition. The second change, a substitution of Gln by Arg in ts118 at residue 93 in nsP4, had little apparent phenotype on its own, but in combination with the change in nsP2 led to a ts phenotype. Thus, in each case the mutation responsible for the temperature sensitivity of the three known complementation group F mutants lay in nsP4. In addition, the result with ts118 suggests that nsP2 and nsP4 may interact with each other in a complex.
|Additional Information:||Copyright © 1989 by the American Society for Microbiology. Received 24 October 1988/Accepted 28 November 1988 The expert technical assistance of E. M. Lenches is gratefully acknowledged. We thank H. V. Huang and S. A. Chervitz for plasmid DNAs. This work was supported by Public Health Service grants AI 10793, AI 20612, and AI 24134 from the National Institutes of Health. C.M.R. is a Pew scholar in the biomedical sciences.|
|PubMed Central ID:||PMC247815|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
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|Deposited On:||11 May 2006|
|Last Modified:||15 Dec 2015 22:26|
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